PROJECT SUMMARY The phosphoribosyl pyrophosphate synthetase (PRPS) enzymes are critical regulators of nucleotide production in all life, from bacteria to man. These enzymes generate a critical precursor necessary for producing all nucleotide species and function as a `molecular throttle' capable of increasing or decreasing the rate at which these genetic building blocks are made. While targeting this metabolic enzyme represents a powerful approach to stymie nucleotide production, the redundancy afforded by the existence of two distinct forms of the same enzyme (PRPS1 and PRPS2) also presents a phenomenal opportunity for selectively treating certain diseases such as cancer. In order to realize this goal, however, we must first have a better understanding of their overlapping and distinct biological roles and the mechanistic basis for these similarities and differences. This proposal seeks to unravel the molecular basis for this selectivity through use of novel mouse models and elegant structure/function studies, thus pinpointing a putative mechanism of action and developing a rational basis for future drug development. We will focus our efforts on elucidating the distinct modes of regulation that control expression of the two separate isoforms as well as how the different biochemical properties of the individual isozymes regulate nucleotide production and metabolic homeostasis. To make our work clinically-relevant and applicable to human disease, we will also develop and characterize novel disease-specific mouse models of PRPS1 superactivity and loss of function that seek to recapitulate the mutations and phenotypes observed in human inborn errors of metabolism. Our research program ultimately strives to understand the complicated role of nucleotides in cellular metabolism and how their aberrant production, breakdown, transport, or utilization contributes to disease. For example, specifically within this proposal, we will elucidate the economics of nucleotide metabolism by determining how disrupting nucleotide supply affects the overall biochemistry of the cell. Collectively, the proposed studies and our research program in general will be transformative in our understanding of the roles of these key molecules in the normal and disease setting, and provide a new foundation for the development of the next generation of safer, more targeted therapies and rational approaches for ameliorating diseases associated with perturbed nucleotide homeostasis.